Immunosuppressive agents

ABSTRACT

A compound with immunosuppressive properties having formula (I), in which R 1  represents a heterocyclic ring system, a combined heterocyclic ring system and homocyclic ring system, or an ethoxycarbonylmethyl group. R 2  and R 3  represent hydrogen or lower alkyl groups.

This is a division of application Ser. No. 08/020,162 filed on Feb. 19, 1993 (U.S. Pat. No. 5,300,519 issued Apr. 5, 1994), which is a continuation of application Ser. No. 07/915,784 filed Jul. 16, 1992 (abandoned), which is a continuation of application Ser. No. 07/663,873 filed Mar. 6, 1991 (abandoned).

This invention relates to immunosuppressive agents.

The purine derivative azathioprine interfers with cell replication, and has immunosuppressive and antileukamic properties. It is used extensively as a drug to treat a wide range of chronic inflammatory diseases in which immune mechanisms are involved such as rheumatoid arthritis; chronic active hepatitis; kidney disease; skin disease; and multiple sclerosis. It is also used to inhibit the rejection of transplanted organs such as kidneys. However, its beneficial therapeutic effects are compromised by the fact that it is toxic to bone marrow. It is the object of the present invention to provide an immunosuppressive agent which does not have this disadvantage.

According to one aspect of the invention there is provided a compound for use as an immunosuppressive agent, said compound having the formula: ##STR1## in which R¹ comprises a heterocyclic ring system, a homocyclic ring system, a combined heterocyclic and homocyclic ring system, alkoxycarbonylalkyl or aryloxycarbonylalkyl group and wherein

R¹ does not comprise ##STR2## or isomers thereof. R² is alkyl, aryl or hydrogen and

R³ is hydrogen, alkyl, aryl or nitroaryl

Preferably

R² is hydrogen, methyl, ethyl or propyl; and

R³ is hydrogen, methyl or 4-nitrophenyl.

According to a preferred embodiment of the invention said compounds which are used as immunosuppressive agents have the following formula: ##STR3## in which R² is hydrogen, or methyl or ethyl or propyl; and

R³ is hydrogen or methyl or 4-nitrophenyl,

and in which R¹ may be one of the following: ##STR4## in which R⁴ is hydrogen or methyl or ethyl or propyl or phenyl or 1-naphthyl ##STR5## in which R⁵ is hydrogen or trifluoromethyl or phenyl or 4-chlorophenyl or 4-methoxyphenyl or 3-pyridyl ##STR6## in which R⁶ is hydrogen or phenyl or chlorophenyl or 4-methoxyphenyl ##STR7## in which R¹⁰ is hydrogen or an alkoxy group; and

R⁷ is hydrogen, alkyl, nitro, halide or an alkoxy group; and

R⁸ is hydrogen, halide or an alkoxy group; and

R⁹ is hydrogen or an alkoxy group ##STR8## in which R¹¹ is hydrogen, methyl, or hydroxyl; and

R¹² is hydrogen or methyl; and

X is nitrogen or C--H ##STR9## in which Z is oxygen or sulphur or N--H; and

Y is C--H or nitrogen ##STR10## in which R¹³ is hydrogen or an amino or carboxyl group; and

R¹⁴ is hydrogen or methyl; and

R¹⁵ is hydrogen or methyl ##STR11## in which R¹⁶ is alkyl or aryl ##STR12## in which

R¹⁷ is alkyl or aryl ##STR13## in which R¹⁸ is alkyl or aryl ##STR14##

Specific illustrative examples of the first embodiment of the invention are as follows: ##STR15##

Some of the compounds, as referred to above are previously unknown.

Therefore, according to a further aspect of the invention there are provided compounds comprising the following formula: ##STR16## wherein the molecular groups R¹, R², and R³ are defined as one of the following:

1.R¹ is defined as the following: ##STR17## in which R⁴ is hydrogen or methyl;

R² is ethyl; and

R³ is methyl

or

R² is methyl and R³ is hydrogen; and

R⁴ is phenyl or 1-naphthyl

or

2. R¹ is defined as the following: ##STR18## in which R², R³, and R⁵ are one of the following combinations:

R² is methyl, R³ is 4-nitrophenyl, and R⁵ is hydrogen; or

R² is methyl, R³ is hydrogen, and R⁵ is trifluoromethyl;

or

R² is ethyl, R³ is methyl, and R⁵ is hydrogen; or

R² is methyl, R³ is hydrogen, and R⁵ is phenyl; or

R² is methyl, R³ is hydrogen, and R⁵ is 4-chlorophenyl; or

R² is methyl, R³ is hydrogen, and R⁵ is 4-methoxyphenyl; or

R² is methyl, R³ is hydrogen, and R⁵ is 3-pyridyl

or

3. R¹ is defined as the following: ##STR19## in which R² is methyl; and

R³ is hydrogen; and

R⁶ is hydrogen or phenyl or 4-chlorophenyl or 4-methoxyphenyl

or

4. R¹ is defined as the following: ##STR20## in which R² is methyl; and

R³ is hydrogen, and

R⁷, R⁸, R⁹, R¹⁰ are one of the following combinations:

R⁸, R⁹, R¹⁰ are all hydrogen and R⁷ is methyl; or

R⁸, R⁹, R¹⁰ are all hydrogen and R⁷ is a nitro group; or

R⁹ and R¹⁰ are both hydrogen, R⁷ is fluorine, and R⁸ is chlorine; or

R⁹ and R¹⁰ are both hydrogen, and R⁷ and R⁸ are both methoxy groups; or

R⁷ and R⁸ are both hydrogen and R⁹ and R¹⁰ are both methoxy groups

or

5. R¹ is defined as the following: ##STR21## in which R² is methyl; and

R³ is hydrogen; and

R¹¹,R¹² and X are defined as one of the following combinations:

R¹¹ and R¹² are both hydrogen, and X is C--H; or

R¹¹ is a hydroxy group, R¹² is hydrogen, and X is nitrogen

or

R² is ethyl; and

R³, R¹¹ and R¹² are all methyl; and

X is nitrogen

or

6. R¹ is defined as the following: ##STR22## in which R² is methyl; and

R³ is hydrogen; and

Y and Z are defined as one of the following combinations:

Y is C--H and Z is oxygen; or

Y is nitrogen and Z is sulphur; or

Y is nitrogen and Z is N--H

or

7. R¹ is defined as the following: ##STR23## in which R² is methyl; and

R³ is hydrogen; and

R¹³, R¹⁴, and R¹⁵ are defined in either of the following combinations:

R¹³ is hydrogen, and R¹⁴ and R¹⁵ are both methyl; or

R¹³ is carboxyl, and R¹⁴ and R¹⁵ are both hydrogen

or

8. R¹ is defined as the following: ##STR24## in which R² is methyl; and

R³ is hydrogen; and

R¹⁶ is methyl or phenyl

or

9. R¹ is defined as the following: ##STR25## or 10. R¹ is defined as the following: ##STR26## or 11. R¹ is defined as the following: ##STR27##

Referring now to Reaction Scheme 1, azathioprine I was originally designed as a "pro-drug" for 6-mercaptopurine II, to which it is rapidly converted by red blood cells. 6-mercaptopurine II has immunosuppressive properties, but the mechanism of its action is unclear.

REACTION SCHEME 1 ##STR28##

It would appear that the immunosuppressive action of azathioprine I is due not solely to prior conversion of 6-mercaptopurine II in the body, but to the immunosuppressive action of azathioprine I itself. The mechanism of action of azathioprine I differs from that of 6-mercaptopurine II. There is strong evidence to suggest that 6-mercaptopurine II is converted in vivo into a nucleotide metabolite that is associated with bone marrow toxicity. There is no evidence to suggest that metabolites derived from the 1-methyl-4-nitroimidazole moiety of azathioprine give rise to toxicity.

It is thought that azathioprine I alkylates thiol groups in the lymphocyte cell membrane, most probably by a process of addition-elimination with the consequent release of 6-mercaptopurine II. It is further supposed that the alkylation of the lymphocyte results in immunosuppression, whilst the 6-mercaptopurine II gives rise, as stated above, via its nucleotide metabolite, to bone marrow toxicity. Support for this hypothesis is provided by the fact that lymphocyte cell membranes are richly endowed with thiol groups, and by the known propensity of azathioprine to react with thiols both in vitro and in vivo.

5-(1-alkyl-4-nitroimidazole) derivatives VI of relatively non toxic alkyl thiols or aryl thiols V are therefore suitable candidates for evaluation as good, relatively non toxic immunosuppressive agents, as is shown in Reaction Scheme 2.

REACTION SCHEME 2 ##STR29##

Examples of immunosuppressive agents of this kind are shown previously in examples 1-43. It is not intended that the invention be limited to these illustrative examples.

Compounds 1 to 17, 19 to 22, 25 to 32, 34 and 36 to 43 may be synthesised, for example, by reaction of 5-chloro-1-methyl-4-nitroimidazole IV with the appropriate thiol V and potassium carbonate in a suitable solvent such as, for example, tetrahydrofuran, dimethylformamide, or water. Compounds 18, 23, 24 and 35 may be synthesised, for example from 5-chloro-1-ethyl-2-methyl-4-nitroimidazole VII and the appropriate thiol under suitable conditions, such as, for example, in acetone in the presence of potassium carbonate.

Sixteen of these compounds were screened for immunosuppressive activity by means of the human mixed lymphocyte reaction, which is well known and need not be further described here, the results of which are shown in Table 1.

                  TABLE 1     ______________________________________                  Concentration                               % Inhibition     Compound     of Solution (μM)                               of .sup.3 M-Thymidine     ______________________________________     I (azathioprine)                  25           79     21           10           89     22           10           82     32           25           98      1           36           98      2           25           86      4           50           59      5           25           94      6           25           41     15           25           76     16           25           47     19           25           29     18           25           47      8           25           87      9           25           27     13           25           34     30           25           65     ______________________________________

The individual figures in the right hand column of the above table are directly proportioned to immunosuppressive activity.

As can be seen from Table 1 all of the compounds screened for immunosuppressive activity displayed significant immunosuppression, and six were substantially more active than azathioprine at the same concentration (25μM).

Table 2 shows the measure of the immunosuppressive activity and toxicity of compounds 21, 22, 32, and 11.

                  TABLE 2     ______________________________________               Immunosuppressive      Therapeutic               activity.sup.a                             Toxicity.sup.b                                      index     Compound  ED.sub.50     LD.sub.25                                      ED.sub.50 /LD.sub.25     ______________________________________     I (azathioprine)               7.9           42.5     5.38     21        2.8           c        vh     22        3.15          d        vh     32        1.6     11        24.0          e        vh     ______________________________________      .sup.a ED.sub.50 = concentration (μM) which brings about 50% inhibitio      of .sup.3 Hthymidine incorporation in the human mixed lymphocyte reaction      lower figures indicate increased reactivity      .sup.b LD.sub.25 = concentration (μM) at which 25% of the cells are      killed: lower figures indicate increased toxicity      .sup.c No LD.sub.25 because only 19% of cells killed at 100 μM      .sup.d No LD.sub.25 because only 15% of cells killed at 215 μM      .sup.e No LD.sub.25 because only 16% of cells killed at 25 μM      (For c, d and e these are maximum values: no more cells were killed at      higher concentrations)      vh very high

Referring now to Table 2, imidazole derivatives 21 and 22 and the triazole derivative 32. are shown to be substantially more immunosuppressive than azathioprine and also considerably less toxic to lymphocytes. Therapeutic indices for these compounds are therefore at least an order of magnitude greater than for azathioprine.

The compound 11 in which the 6-mercaptopurine moiety of azathioprine is replaced by 8-hydroxy-6-mercaptopurine moiety, is less immunosuppressive than azathioprine according to the mixed human lymphocyte reaction, as is shown in Table 2, but is much less toxic to the lymphocytes. The therapeutic index of compound 11 is therefore much greater than that of azathioprine, according to these tests. The significance of this result resides in the fact that 8-hydroxy-6-mercaptopurine VIII is known to be a non toxic metabolite of azathioprine, and its metabolic fate is known.

Compound 32 showed pronounced immunosuppressive activity in vivo. It was tested in CBA mice transplanted with skin from Balb/c mice, according to the skin grafting technique described by Billingham and Medawar (J.Exp. Biol, 28, 385-405 (1951). The effect compound 32 had on skin graft survival was compared with a control of saline, and with the standard immunosuppressive agent azathioprine. All drugs were injected into the peritoneum starting three hours before surgery and then daily until the graft had fully rejected. Mice were placed in groups at random, and rejection of the graft was evaluated by visual inspection by an independent observer. The results are recorded in Table 3.

                  TABLE 3     ______________________________________              Number    Dosage*    Graft survival     Treatment              of mice   (mg/Kg/day)                                   (Mean ± SEM days)     ______________________________________     Control  28        NA         12.5 ± 0.4     Compound 32              24        45         14.2 ± 0.4     athloprine              29        52         12.4 ± 0.3     ______________________________________      *Compound 32 and azathioprine were adminstered in equimolar amounts

The prolongation of graft survival caused by compound 32 (1.7 days) compared to a saline control is significant statistically (p=5.9×10⁻³). Similarly, the prolongation of graft survival caused by compound 32 (1.8 days) compared to azathioprine is also highly significant (p=2.84×10⁻³).

It is to be understood that the above described examples are for illustration only and that many modifications and variations can be made within the scope of the invention. 

We claim:
 1. A method of suppressing the immune system of a human comprising administering to a person requiring same an effective amount of a compound of the following formula: ##STR30## in which R¹ is ##STR31## in which R¹⁰ is hydrogen or an alkoxy group; andR⁷ is hydrogen, alkyl, nitro, halide or an alkoxy group; and R⁸ is hydrogen, halide or an alkoxy group; and R⁹ is hydrogen or an alkoxy group, and R² is a hydrogen, alkyl or aryl; and R³ is a hydrogen, alkyl, aryl or nitroaryl.
 2. A method as claimed in claim 1 in which R² is hydrogen, methyl, ethyl or propyl; and R³ is hydrogen, methyl or 4-nitrophenyl.
 3. A method as claimed in claim 1, having the following formula: ##STR32## in which R² is hydrogen, or methyl or ethyl or propyl, andR³ is hydrogen or methyl or 4-nitrophenyl, and in which R¹ is ##STR33## in which R¹⁰ is hydrogen or an alkoxy group; and R⁷ is hydrogen, alkyl, nitro, halide or an alkoxy group; and R⁸ is hydrogen, halide or an alkoxy group; and R⁹ is hydrogen or an alkoxy group.
 4. A compound comprising the following formula: ##STR34## wherein the molecular groups R¹, R², and R³ are defined as one of the following:R¹ is defined as the following: ##STR35## in which R² is methyl; and R³ is hydrogen; and R⁷, R⁸, R⁹, R¹⁰ are one of the following combinations; R⁹ and R¹⁰ are both hydrogen, and R⁷ and R⁸ are both methoxy groups; or R⁸, R⁹, R¹⁰ are all hydrogen and R⁷ is methyl; or R⁸, R⁹, R¹⁰ are all hydrogen and R⁷ is a nitro group; or R⁹ and R¹⁰ are both hydrogen, R⁷ is fluorine, and R⁸ is chlorine; or R⁷ and R⁸ are both hydrogen and R⁹ and R¹⁰ are both methoxy groups. 